Our long term objectives are to elucidate the questions of how Leishmania infect macrophages, and how these parasites subsequently differentiate, survive and multiply in these phagocytes. Understanding the cellular and molecular mechanisms of such host-parasite interactions in vitro is crucial for developing more effective chemo- and/or immuno-prophylaxis and therapy for leishmaniasis. Leishmania amazonensis molecules previously thought to be important in macrophage infection and thus proposed for investigation are: (1) the major surface glycoprotein/zinc proteinase (gp63); and (2) the 63 kb extrachromosomal DNA circles in tunicamycin (TM)-resistant variants. More emphasis has been placed on the molecular approaches. The gp63 gene of L. amazonensis was cloned and sequenced. Selected residues of gp63 were modified by site-specific mutagenesis of the genes from this and another species. Transfection of gp63-deficient variants with mutant genes allows the identification of catalytically and parasitically important residues. Two additional genes were identified in the 63 kb DNA circle by functional analysis after transfection of wildtype cells with deletion clones of this DNA. One was found to confer tunicamycin-resistance, apparently encoding N-acetylglucosamine-1- phosphate transferase (NAGTase). Using this gene as a selective marker, another gene immediately upstream was identified and found to overexpress a 36 kd protein, homologous to zeta-crystallin/NADPH quinone oxidoreductase (P36). It is proposed to continue the above-mentioned work in conjunction with the established in vitro transfection and macrophage infection systems as follows: (1) To optimize the efficiency of transfection for functional analysis of gp63 and other relevant molecules; (2) To further analyze the biological importance of catalytic, receptor-binding and other functional motifs of gp63 in leishmanial infection; (3) To further characterize the functional significance of posttranslational modifications in regulating gp63 expression by using N-glycosylation and GPI-anchor mutants; (4) Attempts to identify additional molecules of relevance by negative and positive phenotype selections; (5) To delineate "avirulent" promastigote- derived axenic amastigotes as mutants defective in intracellular differentiation and/or replication. The results of these proposed studies will help our understanding not only Leishmania molecules in their infection of macrophages but also comparable molecules in other biological systems.